Figures

Abstract

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-α, IFN-β and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-λ uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR10/0) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-λ might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-λ readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR10/0 mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-λ failed to induce Mx1 in the liver of IFNAR10/0 mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-λ receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-α/β and IFN-λ were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR10/0 mice. From these results we conclude that IFN-λ contributes to inborn resistance against viral pathogens infecting the lung but not the liver.

Author Summary

The contribution of IFN-λ to innate immunity against virus-induced diseases has remained unclear to date as appropriate mouse models were not available. We now present evidence that IFN-λ is involved in the antiviral defense. Mice lacking functional IFN-λ receptors were only slightly more susceptible to influenza virus than wild-type mice, but intranasal administration of IFN-λ efficiently protected IFN-α/β receptor-deficient mice from lethal influenza virus infection and induced the antiviral factor Mx1 in lungs. Mice lacking functional receptors for both IFN-α/β and IFN-λ were hypersensitive and failed to restrict even usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. By contrast, intraperitoneal application of IFN-λ failed to induce Mx1 in the liver of mice and did not protect against hepatotropic viruses. Furthermore, double-knockout mice were not more susceptible against hepatotropic viruses than IFN-α/β receptor-deficient mice, indicating that IFN-λ contributes to resistance against viral pathogens infecting the lung but not the liver.

Funding: This work was supported by grant STA 338/7-1 from the Deutsche Forschungsgemeinschaft (DFG) to P.S. and G.K.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Viral infection of vertebrate cells triggers innate immune responses, which result in rapid synthesis of IFN and other pro-inflammatory cytokines [1]–[4]. Virus-induced IFN represents a complex mixture of IFN subtypes which act on target cells by engaging two distinct cell surface receptors [5]. All members of the type I IFN family which, in the mouse, includes 14 different IFN-α subtypes, IFN-β, IFN-κ, IFN-ε and limitin, use the same heterodimeric IFN-α/β receptor complex (IFNAR1/2) for signaling [6]. By contrast, signaling by type III IFN family members (in the mouse IFN-λ2 and IFN-λ3) occurs through the heterodimeric interleukin-28 receptor α/interleukin-10 receptor β (IL-28Rα/IL-10Rβ) complex [7],[8]. Although activating distinct receptor systems, IFN-λ and type I IFNs trigger strikingly similar responses in target cells which mostly result from phosphorylation-induced activation of transcription factors STAT-1 and STAT-2 [9],[10]. The IFNAR1/2 complex is present on most if not all nucleated cells, whereas expression of the IL-28Rα subunit seems to be cell type-restricted [11],[12]. Consequently, antiviral protection by type I IFN is observed in most cell types, whereas antiviral protection mediated by IFN-λ is restricted to cells that express functional IL-28R complexes. The spectrum of cell types that respond to IFN-λ in vivo is poorly defined. Recent experiments suggested that epithelial cells are the main targets of IFN-λ in the mouse [13].

Information on the contribution of IFN-λ to virus resistance at the level of the whole organism is very limited as mice lacking functional IFN-λ receptors (IL28Rα0/0) were generated only recently [14]. Unlike knockout mice lacking functional type I IFN receptors (IFNAR10/0) that are highly susceptible to a broad spectrum of different viruses [15], IL28Rα0/0 and wild-type mice did not differ significantly in resistance to a large panel of pathogenic viruses [14]. The only observed difference between wild-type and IL28Rα0/0 mice was that treatment of knockout mice with toll-like receptor (TLR) 3 and TLR9 agonists failed to induce resistance to vaginal infection with herpes simplex virus type 2 [14].

Here we used Mx1+/+ mice to investigate the relative contributions of IFN-λ and type I IFN in immunity toward influenza A virus. Mx1+/+ mice differ from standard mouse strains in being fully IFN-competent. They carry functional alleles of the influenza virus resistance gene Mx1, which is defective in standard laboratory mice [16]. Consequently, in Mx1+/+ mice, virus-induced IFN activates the Mx1 gene in addition to other antiviral genes, leading to a more complete innate immune response and more robust resistance to influenza and influenza-like viruses [17],[18]. The Mx1+/+ mouse model system has the power to reveal even subtle defects in antiviral immunity against orthomyxoviruses. It has recently been used to uncover the beneficial effect of IFN-β in influenza virus defense [19]. It was further used to demonstrate that IFN-α might be used to prevent disease induced by highly lethal human H5N1 influenza viruses [17]. Using this experimental system we now demonstrate that IFN-λ contributes to innate immunity against influenza virus but not against two different hepatotropic viruses. These differences in virus susceptibility correlated with the differing ability of virus-induced IFN-λ to activate the Mx1 gene in lung and liver of IFNAR10/0 mice.

Results

IFN-λ is induced in virus-infected lung and liver of IFNAR10/0 mice

Since virus-induced activation of IFN genes requires positive feedback through the IFN-α/β receptor in certain cell types [20], we first determined whether the major IFN subtypes are induced in lung and liver of IFNAR10/0 mice after infection with viruses that strongly activate the innate immune system. As can be seen in Figure 1, we observed strong transcriptional activation of genes for the IFN-α family, IFN-β and IFN-λ2 in the lung of mice infected intranasally with the influenza A virus mutants SC35M-ΔNS1 and PR8-ΔNS1 that are known to induce large amounts of type I IFN [21],[22]. Similarly, strong transcriptional activation of IFN-α, IFN-β and IFN-λ genes was observed in the liver of IFNAR10/0 mice infected with a mutant of hepatotropic Thogotovirus (THOV) that lacks the IFN-antagonistic factor ML (THOV-ΔML) [23].

Animals were either infected by the intranasal route with 106 pfu of influenza A virus strain SC35M-ΔNS1 or PR8-ΔNS1, or else by the intraperitoneal route with 10 pfu of hepatotropic THOV-ΔML. Animals treated with plain buffer served as negative controls. At 17 hours post infection, the influenza virus-infected mice were killed and the lungs were removed for analysis. The liver of the THOV-infected mouse was harvested when the animal was severely diseased at 72 hours post infection. RNA samples from the organs were reverse transcribed and analyzed by PCR for transcripts of the indicated genes.

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Exogenous IFN-λ protects IFNAR10/0 mice against intranasal challenge with influenza A virus but not against intraperitoneal challenge with Thogotovirus

In a first experiment, IFNAR10/0 mice were treated with exogenous IFN-λ by the intranasal route to determine whether this cytokine might contribute to protection from pneumonia induced by pathogenic influenza viruses. Groups of IFNAR10/0 mice were treated with 7,500 units of either recombinant IFN-λ2 or IFN-λ3. Control animals received corresponding volumes of a mock preparation. Ten hours later, the animals were infected with 100 plaque-forming units (pfu) (~20 LD50) of mouse-adapted H7N7 influenza A virus strain SC35M [22]. The control animals quickly lost weight and had to be killed between days 7 and 9 post infection due to clinical signs of pneumonia, whereas all animals treated with either IFN-λ2 or IFN-λ3 remained healthy (Fig. 2A). Since standard IFNAR10/0 mice lacking functional Mx1 alleles developed severe disease under identical experimental conditions in spite of treatment with IFN-λ3 (data not shown), we concluded that the protective effect of IFN-λ that we observed in our Mx1+/+ mice was mainly mediated by the IFN-induced resistance factor Mx1.

Figure 2. Exogenous IFN-λ protects IFNAR10/0 mice against intranasal challenge with influenza A virus but not against intraperitoneal challenge with THOV.

(A) Survival of mice intranasally treated for 10 hours with a mock preparation or 7,500 units of either IFN-λ2 or IFN-λ3 before challenge with 100 pfu (~20 LD50) of influenza A virus strain SC35M. (B) Survival of mice intraperitoneally treated for 10 hours with a mock preparation or 15,000 units of IFN-λ3 before infection with 100 pfu (~20 LD50) of THOV.

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In a second experiment, 15,000 units of IFN-λ3 were applied by the intraperitoneal route to IFNAR10/0 mice carrying functional Mx1 alleles. Ten hours later the animals were challenged with 100 pfu (~20 LD50) of THOV. Animals treated with IFN-λ3 as well as control animals treated with a mock preparation developed severe disease between 48 and 96 hours post infection (Fig. 2B). Thus, IFN-λ exhibited effective antiviral activity in the lung, but seemed to be inactive in the liver.

IFN-λ activates Mx1 gene expression in the lung but not liver of IFNAR10/0 mice

Like type I IFN, IFN-λ exhibits antiviral activity by binding to a specific cell receptor complex that can activate latent STAT transcription factors [24]. After activation, the STAT proteins move to the nucleus where they activate transcription of a large number of IFN-responsive genes, including Mx1. To determine whether exogenous IFN-λ activates IFN-responsive genes in our IFNAR10/0 mice, we harvested lung and liver at 20 hours post onset of treatment with IFN-λ3 and analyzed the tissue homogenates for Mx1 protein by western blotting. Easily detectable levels of Mx1 were present in lungs of mice that were treated intranasally with 3,500 units of IFN-λ3 (Fig. 3A). The lungs of mock-treated mice did not contain detectable levels of Mx1.

(A) Western blot analysis of Mx1 protein levels in lungs of mice at 20 hours post intranasal application of 3,500 units of IFN-λ3. Animals treated with a mock preparation served as control. Two animals of each group are shown. (B) Mx1 protein levels in the liver of IFNAR10/0 mice at 20 hours post intraperitoneal application of 15,000 units of IFN-λ3 or terminally ill at 72 hours post infection with hepatotropic THOV-ΔML or RVFV clone 13. Two animals for each group are shown. Liver extract from a wild-type mouse killed at 20 hours post intraperitoneal treatment with 100,000 units of human IFN-αB/D served as positive control.

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Mx1 protein could not be detected in the liver of IFNAR10/0 mice treated with 15,000 units of IFN-λ3 by the intraperitoneal route (Fig. 3B). If, as a control, a cross-reactive variant of human IFN-α was injected by the same route into wild-type mice, Mx1 was prominently induced in the liver (Fig. 3B). To distinguish between the possibility that liver cells lack functional receptors for IFN-λ and the possibility that the recombinant IFN-λ failed to reach the liver under our experimental conditions, we analyzed the Mx1 protein levels in IFNAR10/0 mice infected with THOV-ΔML which strongly induces IFN-λ in the liver (Fig. 1). The liver of mice with severe THOV-induced disease contained no detectable amounts of Mx1 protein (Fig. 3B). Similarly, no Mx1 protein could be detected in the liver of terminally ill IFNAR10/0 mice infected with Rift Valley fever virus clone 13 (Fig. 3B), another hepatotropic virus with strong IFN-inducing activity [25]. Thus, differential IFN-λ receptor expression in lung and liver seemed to explain why exogenously applied IFN-λ protected IFNAR10/0 mice from virus-induced disease of the lung but not the liver.

To directly assess the contribution of IFN-λ to the protection from influenza virus-induced lung disease, we generated IL28Rα0/0 mice carrying functional Mx1 alleles by crossbreeding of appropriate mouse strains and compared the fate of wild-type and IL28Rα0/0 mice after challenge with 5×104 plaque-forming units (pfu) of SC35M. Survival of IL28Rα0/0 mice was slightly reduced compared to wild-type mice (Fig. 4A), but the difference was not statistically significant. Viral titers in lungs of IL28Rα0/0 mice were slightly but significantly higher at 72 h post infection than in lungs of wild-type mice (Fig. 4B).

Wild-type and IL28Rα0/0 mice were infected by the intranasal route with 5×104 pfu of SC35M. (A) Survival and (B) virus titers in lungs at 72 hours post infection were recorded. Combined data of several independent experiments are shown. (**: p<0.01).

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Strongly reduced resistance to influenza virus but not to hepatotropic viruses of IFNAR10/0 mice lacking functional receptors for IFN-λ

To determine the relative contributions of IFN-α/β and IFN-λ in antiviral defense we generated Mx1+/+ mice that lack functional receptors for both of these two classes of IFN (IFNAR10/0IL28Rα0/0) and compared them to mice that lack receptors for IFN-α/β only. We previously demonstrated that IFNAR10/0 mice with intact Mx1 alleles are highly susceptible to challenge infections with wild-type SC35M [19]. However, intranasal infection with 105 pfu of SC35M-ΔNS1 did not induce disease in IFNAR10/0 mice (Fig. 5A). Similarly, all wild-type and IL28Rα0/0 mice remained healthy when challenged with up to 106 pfu of SC35M-ΔNS1 (data not shown). In marked contrast, all IFNAR10/0IL28Rα0/0 double-knockout mice infected with 105 pfu of SC35M-ΔNS1 developed severe disease and had to be killed around day 5 post infection (Fig. 5A). Additional experiments in which we used lower doses of challenge virus demonstrated that the LD50 of SC35M-ΔNS1 in IFNAR10/0IL28Rα0/0 double-knockout mice was approximately 103 pfu (Fig. 5A). A similar picture emerged when the mice were challenged with a NS1-deficient variant of the H1N1 human influenza A virus strain PR8 (PR8-ΔNS1). At a dose of 106 pfu, all infected IFNAR10/0IL28Rα0/0 double-knockout mice developed severe pneumonia within 4–6 days post infection, whereas all IFNAR10/0 single-knockout mice remained healthy (Fig. 5B). Importantly, our single- and double-knockout mice did not differ in susceptibility to infection with the two hepatotropic viruses THOV-ΔML (Fig. 5C) and RVFV clone 13 (Fig. 5D), strongly supporting the above-formulated conclusion that IFN-λ is not active in the liver of IFNAR10/0 mice.

Survival of IFNAR10/0 (diamonds) and IFNAR10/0IL28Rα0/0 double knockout mice (circles) after (A) intranasal infection with the indicated doses of SC35M-ΔNS1, (B) intranasal infection with 106 pfu of PR8-ΔNS1, (C) intraperitoneal infection with the indicated doses of THOV-ΔML, and (D) intraperitoneal infection with the indicated doses of RVFV clone 13. Groups consisted of four to nine animals.

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High virus load in lungs of mice correlates with low Mx1 protein levels

Virus replication in lungs of wild-type and mutant mice was assessed at 48 hours post infection with 105 pfu of SC35M-ΔNS1. Virus titers in lungs of wild-type mice were below the detection limit in four of five animals, and they were only slightly above the detection limit in lungs of IL28Rα0/0 mice at 48 h post infection (Fig. 6A). Remarkably, SC35M-ΔNS1 did not grow much better in lungs of IFNAR10/0 mice, whereas it replicated to very high titers in lungs of IFNAR10/0IL28Rα0/0 double-knockout mice (Fig. 6A). At 20 hours post infection with SC35M-ΔNS1 the Mx1 protein levels in lungs of IL28Rα0/0 mice were about 2-fold lower than in the wild-type animals (Fig. 6B). Lungs of infected IFNAR10/0 mice contained about 10-fold lower levels of Mx1 protein than wild-type mice, whereas Mx1 levels were below the detection limit in IFNAR10/0IL28Rα0/0 double-knockout mice (Fig. 6B). Thus, after infection with SC35M-ΔNS1, the extent of Mx1 gene expression in lungs of mice with defective receptors for IFN-α/β, IFN-λ or both correlated inversely with virus titers.

Groups of mice were infected with 105 pfu of SC35M-ΔNS1 and either killed at (A) 48 hours post infection to determine viral titers in the lung or at (B) 20 hours post infection to determine Mx1 protein levels by western blotting. Two animals of each group are shown. Actin-normalized Mx1 signal intensities are indicated. The calculated value of the wild-type mice was set to 100%. (*: p<0.05), ***: p<0.001).

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Discussion

The intracellular signaling pathways activated by IFN-λ and IFN-α/β are quite similar [9],[10], suggesting that both IFN types are contributing to virus resistance. Surprisingly, however, mice lacking functional receptors for IFN-λ did not differ from wild-type mice when challenged with a panel of different pathogenic viruses [14]. A mild deficiency of IFN-λ-deficient mice became only apparent in an experimental setting in which resistance to herpes simplex virus type 2 was induced by treating the animals with TLR3 or TLR9 agonists [14]. This phenotype is in marked contrast to that of mice lacking functional receptors for IFN-α/β which are highly susceptible to many viruses [15].

We reasoned that the different phenotypes of the knockout mice might be explained by the different expression patterns of the receptors for IFN-α/β and IFN-λ in the organism. Receptors for IFN-α/β are rather uniformly expressed on most if not all nucleated cells [26], whereas receptors for IFN-λ are preferentially expressed on epithelial cells [13]. If our reasoning was correct, one would predict that the protective effect of IFN-λ should be restricted to organs with a high percentage of cells expressing the IFN-λ receptor and that the protective effect of IFN-λ in these organs might be most obvious when the IFN-α/β system is defective. In this report we provide evidence that strongly supports this view.

We observed that intranasal application of IFN-λ protected the mice from lethal challenge with influenza A virus, whereas systemic application of IFN-λ failed to mediate protection from disease induced by a hepatotropic virus (Fig. 2). It should be noted that the mice employed here lacked functional IFN-α/β receptors, excluding the possibility that the protective effect in the lung was indirect and resulted from IFN-α/β that might have been induced by undefined contaminating substances in our IFN-λ preparations. Protection against influenza virus correlated with the presence of the IFN-induced Mx1 protein in the lung tissue (Fig. 3), suggesting that lung epithelial cells carry functional IFN-λ receptors. By contrast, no Mx1 protein was found in liver tissue of mice treated with IFN-λ (Fig. 3). The liver tissue also failed to respond to IFN-λ synthesized in the virus-infected liver (Fig. 1), suggesting that mouse liver cells do not express functional receptors for IFN-λ. This latter conclusion is in agreement with results from recent quantitative RT-PCR analyses which showed that the alpha chain of the IFN-λ receptor (IL28R-α) is expressed only at very low levels in liver of mice [13]. However, our results appear to be in conflict with a previous report in which IFN-λ was successfully used to inhibit hepatitis B virus replication in a murine hepatocyte cell line expressing the viral genome as a transgene [27]. However, these authors observed no induction of IFN-responsive genes in the liver of mice treated with large amounts of IFN-λ, and they observed no inhibition of hepatitis B virus replication in vivo[27]. In this respect, hepatocyte cell lines may not mirror the normal behavior of hepatocytes in intact liver tissue.

Since the virus challenge studies in a former report [14] were carried out with IFN-λ receptor knockout mice lacking the IFN-induced influenza virus resistance factor Mx1, it remained possible that the beneficial effect of IFN-λ against influenza virus had previously been underestimated. Yet, our new experiments with Mx1-positive mice revealed that the lack of IFN-λ system has indeed a much less drastic effect on virus resistance than the lack of the IFN-α/β system. The protective role of IFN-λ became only apparent in Mx1-positive mice that lack a functional IFN-α/β system, and it was most prominent if influenza virus mutants with high IFN-inducing potential were used (Fig. 5). It is well known that highly pathogenic influenza viruses are not controlled well by the IFN system because the virus-encoded NS1 protein counteracts efficient activation of IFN genes in infected cells [21]. NS1-deficient influenza viruses which are very potent IFN inducers are highly attenuated in wild-type mice but remain virulent in mice lacking STAT-1 [21], a transcription factor centrally placed in the signaling pathways of all IFN types [28]. We found that mutants of the influenza virus strains SC35M and PR8 lacking NS1 were completely non-virulent in IFN-α/β receptor-deficient mice and failed to replicate efficiently in the lung of such mice (Figs 5A and 5B), which should not be the case if IFN-α/β was the only IFN subtype that confers resistance to influenza viruses. Our observation that double knockout mice lacking functional receptors for IFN-α/β and IFN-λ are highly susceptible to the NS1-deficient influenza virus mutants clearly demonstrates that IFN-λ provides the residual protection in IFN-α/β receptor-deficient mice.

Some important conclusions can be drawn from our data regarding the role of different IFN types in antiviral immunity. First, the virus defense strategy of the lung is not exclusively based on the IFN-α/β system. Our data clearly demonstrate that the IFN-λ system also contributes to innate immunity against influenza A virus. The second important conclusion from our study is that the IFN-α/β system is dominant over the IFN-λ system. IFN-λ thus appears to be part of a secondary defense system which can fill gaps left by the IFN-α/β system. Future studies will help to distinguish between the possibility that IFN-λ is predominantly active against influenza viruses and the possibility that IFN-λ plays a broader role in the lung and improves innate immunity against other pathogenic viruses that infect the respiratory tract. Evidence in favor of the second possibility includes the observation that IFN-λ also restricted vaccinia virus replication in the lung of mice [29]. We further noted with interest that, reminiscent to the situation with NS1-deficient influenza virus, IFN-α/β receptor-deficient mice are able to restrict the growth of human respiratory syncytial virus in the lung far better than STAT-1-deficient mice [30]. This observation suggests that IFN-λ might also help controlling respiratory syncytial virus. Since receptors for IFN-λ are expressed on epithelial cells of many different organs including lung, stomach and intestine [13], it is conceivable that the physiological role of this cytokine is to protect the host from viral infections via mucosal membranes at many different body sites. An important issue to be addressed in the future is whether IFN-λ might serve a similar role in humans.

Materials and Methods

Mice

All animals used were of C57BL/6 genetic background. Congenic B6.A2G-Mx1 mice [31] carrying intact Mx1 alleles and B6.A2G-Mx1-IFNAR10/0 mice lacking functional type I IFN receptors [19] were bred locally. C57BL/6 mice lacking functional type III IFN receptors (IL28Rα0/0) [14] were crossed with B6.A2G-Mx1 and B6.A2G-Mx1-IFNAR10/0 mice to produce strains with intact Mx1 alleles and defective alleles for IL28Rα only, or IL28Rα and IFNAR1 in combination. Six- to eight-week-old animals were used for all infection experiments, which were performed in accordance with the guidelines of the local animal care committee. Animals were euthanized if severe symptoms developed or body weight loss approached 30% of the initial value.

Viruses

We used wild-type influenza A virus strains SC35M (H7N7) and A/PR/8/34 (H1N1) as well as mutants SC35M-ΔNS1 [22] and PR8-ΔNS1 [21] lacking the IFN-antagonistic factor NS1. We further used wild-type Thogotovirus (THOV) or mutant THOV-ΔML lacking the IFN-antagonistic factor ML [23], and the attenuated “clone 13” strain of Rift Valley fever virus (RVFV) lacking functional IFN-antagonistic factor NSs [25]. All these viruses are classified as BSL2 pathogens in Germany.

Virus infections

Animals were anesthetized by intraperitoneal injection of a mixture of ketamine (100 µg per gram body weight) and xylazine (5 µg per gram body weight) before intranasal infection with the indicated doses of the various influenza A viruses in 50 µl PBS containing 0.3% BSA. For THOV and RVFV infections, 100 µl-samples of diluted virus stocks were applied intraperitoneally without anaesthesia.

Cytokines

IFN-λ2 and IFN-λ3 were produced by transient transfection of 293T cells with appropriate expression plasmids [9]. The biological activity of IFN-λ2 and IFN-λ3 was determined as previously described [32]. Hybrid human IFN-αB/D that is highly active on mouse cells was used as positive control [17],[33].

IFN treatment of mice

Samples containing the indicated amounts of IFN-λ2 or IFN-λ3 were either applied intranasally (50 µl) to anesthetized animals or injected intraperitoneally (200–300 µl) without anaesthesia.

Titration of virus in lungs

Lung homogenates were prepared by grinding the tissue using a mortar and sterile quartz sand. Homogenates were suspended in 1 ml of PBS, and tissue debris was removed by low speed centrifugation. Virus titers in supernatants were determined by performing plaque assays on MDCK II cells by serial 10-fold dilutions in PBS containing 0.3% BSA.

RT-PCR

Lung and liver were removed, and frozen immediately in liquid nitrogen. RNA was isolated from the organs using 1 ml of TriFast according to the protocol of the manufacturer (peQLab). The RNA was further purified by using RNeasy mini kit columns (Qiagen). One µg of each RNA preparation was reverse-transcribed using random-hexamer primers and reverse transcriptase. The reaction products were used to amplify the cDNAs by Taq polymerase for 30 cycles using the indicated primer pairs for mouse IFN-β (accession no. NM_010510, primers from positions 21–42 and 145–124), the mouse IFN-α family (accession no. NM_010504, primers from positions 46–68 and 557–534), mouse IFN-λ2 (accession no. NM_001024673, primers from positions 83–104 and 191–170), and mouse β-actin (accession no. X03672, primers from positions 1374–1396 and 1585–1564). RT-PCR products were separated by agarose electrophoresis, stained with ethidium bromide and visualized under UV light.